Photoactivated crosslinking of a protein or peptide

ABSTRACT

A method of crosslinking a protein or peptide for use as a biomaterial, the method comprising the step of irradiating a photoactivatable metal-ligand complex and an electron acceptor in the presence of the protein or peptide, thereby initiating a cross-linking reaction to form a 3-dimensional matrix of the biomaterial.

TECHNICAL FIELD

The present invention relates to photoactivated crosslinking of aprotein or peptide to form a biomaterial and, more particularly, to thepreparation of a manufactured article of cross-linked proteinaceousmaterial, the manufacture of such materials and their uses. Withoutlimitation, in embodiments the invention relates to a peptidic orproteinaceous scaffold for tissue engineering and methods for the use ofsuch a scaffold. The present invention additionally relates to a methodof adhesion or the joining and/or sealing tissues involvingadministration of a photoactivatable composition in surgical proceduresand medical methods, and compositions for use in said methods. Thepresent invention additionally relates to a method of joining and/orsealing non-biological materials and compositions for use for thispurpose.

BACKGROUND ART

Tissue engineering including the use of biomaterials offers a novelroute for repairing damaged or diseased tissues by incorporating thepatients' own healthy cells or donated cells into temporary housings orscaffolds as well as sealing and/or joining severed tissues. Thestructure and properties of the scaffold are critical to ensure normalcell behaviour and performance of the cultivated or repaired tissue.Biomaterials play a crucial role in such schemes by offering flexibledesign opportunities, directing subsequent cellular behaviour orfunction, as well as facilitating resorption rates and ultimate tissueform and strength.

A range of approaches has been used for the construction and assembly ofsuch biomaterials, including the use of a number of synthetic materials,but it is clear that materials from natural sources are superior becauseof their inherent properties of biological recognition, and theirsusceptibility to cell-triggered proteolytic breakdown and remodelling.

Natural protein such as extracellular matrix (ECM) proteins show promisein tissue engineering applications because of their biocompatibility,but have been found to be lacking in many areas as a result ofinappropriate physical properties. For example, McManus et al (2006)have found that electrospun fibrinogen has insufficient structuralintegrity for implantation, and instead employed an electrospunfibrinogen-polydioxanone (PDS) composite scaffold for urinary tractreconstruction. Fibrinogen, collagen, elastin, haemoglobin andmyogloglobin are reported to have been electrospun (Barnes et al, 2006).The electrospinning process involves imparting a charge to a polymersolution (or melt) and drawing the charged solution into a nozzle. Asthe electrostatic charges within the solution overcome the surfacetension, a liquid jet is initiated at the nozzle. The liquid jet isdirected to a rotating mandrel some distance away. As the solutiontravels the solvent evaporates, and a film is deposited on the mandrel,thus a non-woven, fibrous mat is produced. Additionally, fibrinmicrobeads and nanoparticles are described in WO 03/037248 (HaptoBiotech, Inc.) and comprise beads of fibrinogen and thrombinmanufactured by mixing an aqueous solution of fibrinogen, thrombin andFactor XIII and oil at 50-80C to form an emulsion. To form nanoparticlesthe emulsion so-formed is homogenised and the nanoparticles isolated byfiltration as a fibrin clot created following cleavage of fibrinogenunder the influence of thrombin and Factor VIII. However, these beadsand fibres are limited in their shape configuration and flexibility.

Biomaterials such as tissue adhesives have been suggested asalternatives in surgical procedures to physical procedures of connectingtissues such as sutures and staples. Tissue adhesives will hold cut orseparated areas of tissue together to allow healing and/or serve as abarrier to leakage, depending on the application. The adhesive shouldbreak down or be resorbed and it should not hinder the progress of thenatural healing process. Ideally, the agent should promote the naturalmechanism of wound healing and then degrade.

Tissue adhesives are generally utilized in three categories:

i) Hemostasis (for example, by improving in vivo coagulation systems,tissue adhesion itself has a hemostatic aim and it is related to patientclotting mechanisms)

ii) Tissue sealing: primary aim is to prevent leaks of varioussubstances, such as air or lymphatic fluids.

iii) Local delivery of exogenous substances such as medications, growthfactors, and cell lines.

One accepted value of fibrin glues lies in their unique physiologicaction, which mimics the early stages of the blood coagulation processand wound healing; the part of the normal coagulation cascade to producean insoluble fibrin matrix. Fibrinogen is a plasma protein which isnaturally cleaved to soluble fibrin monomers by the action of activatedthrombin. These monomers are cross-linked into an insoluble fibrinmatrix with the aid of activated factor XIII. The adhesive qualities ofconsolidated fibrin sealant to the tissue may be explained in terms ofcovalent bonds between fibrin and collagen, or fibrin, fibronectin andcollagen. Fibrin glues act as both a hemostatic agent and as a sealant.They are bioabsorbable (due to in vivo thrombolysis). Degeneration andreabsorption of the resulting fibrin clot is achieved during normalwound healing.

All fibrin sealants in use as of 2008 have multi-component having twomajor ingredients, fibrinogen and thrombin and optionally human bloodfactor XIII and a substance called aprotinin, which is derived fromcows' lungs. Factor XIII is a compound that strengthens blood clots byforming covalent cross-links between strands of fibrin. Aprotinin is aprotein that inhibits the enzymes that break down blood clots. Howeverthese sealants being multicomponent require double barrelled syringes,reconstitution of the multiple components and require exquisite mixingduring application to give rise to a uniform and efficious glue.

In an effort to develop a single component protein derived biomaterial,purified thrombin has been developed and now marketed to controllingbleeding during surgery. Upon its application to the tissue site thethrombin cleaves endogenous fibrinogen to produce fibrin in vivo. It iswell known that fibrin (which forms the fibrillar matrix on thrombincleavage of fibrinogen) self-associates (Mosesson M W (2005) Mosesson etal M W, 2001). Factor XIII may be co-administered, and causesdimerisation of the γ-chain of fibrinogen in association with itscleavage by thrombin (Furst W, et al (2007). The success of theprocedure relies upon Factor XIII-mediated crosslinking (Lee M G andJones D (2005) to stabilise the thrombin-derived clot, and a process ofstabilising the clot which does not rely on the presence of Factor XIIIwould be desirable. This single component biomaterial is limited in itsapplicability, can practically only be used for small bleeds, and theresultant clot, which is slow to form typically has low mechanicalstrength.

Despite the availability of all of these different biomaterials for thesurgeon to use in various surgical procedures there still remains a needfor a simple biomaterial that is tunable in its mechanical andbiological properties, is easy to use and apply and can be used in avariety of applications for a variety of diseases and surgicalprocedures.

SUMMARY OF THE INVENTION

In one aspect there is provided a method of crosslinking a protein orpeptide for use as a biomaterial, the method comprising the step ofirradiating a photoactivatable metal-ligand complex and an electronacceptor in the presence of the protein or peptide, thereby initiating across-linking reaction to form a 3-dimensional matrix of thebiomaterial.

In a further aspect there is provided a biomaterial comprising a3-dimensional matrix of a protein or peptide crosslinked throughirradiation a photoactivatable metal-ligand complex and an electronacceptor in the presence of the protein or peptide, thereby initiating across-linking reaction to form a 3-dimensional matrix of thebiomaterial.

In a still further aspect there is provided a method of joining and/orsealing tissues in a surgical procedure or medical treatment, comprisingthe steps of:

(1) applying to a tissue portion a photoactivatable metal-ligand complexand an electron acceptor and optionally an at least partially denaturedprotein

(2) irradiating said tissue portion to photoactivate thephotoactivatable metal-ligand complex;

thereby initiating a cross-linking reaction between

(a) one or more endogenous proteins and/or

(b) said at least partially denatured protein

to seal said tissue portion or join said tissue portion to an adjacenttissue portion and

wherein said at least partially denatured protein has been rendered moresusceptible to photochemical cross-linking compared to its native state

In a still further aspect there is provided a closure for a leakingwound comprising a substrate suitable for application to a wound to stemleakage, wherein said substrate is impregnated or coated with aphotoactivatable metal-ligand complex and an electron acceptor or withan at least partially denatured protein, a photoactivatable metal-ligandcomplex and an electron acceptor, wherein said at least partiallydenatured protein has been rendered more susceptible to photochemicalcross-linking compared to its native state.

In a yet another aspect there is provided the use of thrombin, aphotoactivatable metal-ligand complex and an electron acceptor forjoining and/or sealing tissues.

In a yet another aspect there is provided the use of a photoactivatablecomplex and an electron acceptor for joining and/or sealing tissues.

In a yet another aspect there is provided a composition comprising an atleast partially denatured protein, a photoactivatable metal-ligandcomplex and an electron acceptor, wherein said at least partiallydenatured protein or chemically modified protein has been rendered moresusceptible to photochemical cross-linking compared to its native state.

In a yet another aspect there is provided the use of an at leastpartially—denatured protein, a photoactivatable metal-ligand complex andan electron acceptor for joining and/or sealing tissues.

In a yet another aspect there is provided the use of a protein orpeptide, a photoactivatable metal-ligand complex and an electronacceptor for joining and/or sealing substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a photograph of electrophoresis gel in which reactionmixtures containing 25 μg of bovine fibrinogen (Sigma); 2 mM[Ru(bpy)₃]Cl₂; 20 mM persulfate (Sodium salt) all in 25 μl PBS wereexposed to 300 W incoherent light from Quartz Halogen dichroic sourcefor various times:

Lane No. Sample 1  0 secs 2  1 sec 3  2 sec 4  5 sec 5 10 sec 6 30 sec 760 sec 8 Protein size standards

FIG. 2 shows a photograph of an electrophoresis gel in which reactionsmixtures containing 25 μg of bovine fibrinogen (Sigma); 20 mM persulfate(Sodium salt) and various concentrations of [Ru(bpy)₃]Cl₂, all in 25 μlPBS, were exposed to 300 W incoherent light from Quartz Halogen dichroicsource for 1 min.

Lane No. Sample 1. MW Standards (as above) 2. 2 mM [Ru(bpy)₃]Cl₂, NoLight 3. 0 [Ru(bPY)₃]Cl₂ 4. 0 NaPS 5. 1 μM 6.   5 ″ 7.  10 ″ 8.  25 ″ 9. 50 ″ 10.  100 ″ 11.  500 ″ 12. 2000 ″

FIG. 3 shows a photograph of an electrophoresis gel in which reactionmixtures containing 25 μg of bovine fibrinogen (Sigma); 2 mM[Ru(bpy)₃]Cl₂ (Aldrich) all in 25 μl PBS. (SPS: sodium persulfate; APS:ammonium persulfate) were exposed to 300 W incoherent light from QuartzHalogen dichroic source for 1 min.

Lane No. Sample 1.   20 mM SPS 2.   10 mM ″ 3.   5 mM ″ 4.  2.5 mM ″ 5.1.25 mM ″ 6. 0.63 mM ″ 7. 0.31 mM ″ 8.   10 mM APS 9.  2.5 mM ″ 10. 0.63mM ″ 11.   0 persulfate 12. MW Standards. (as above)

FIG. 4 is an electrophoresis gel showing the results of cross-linkingwhen alternative electron acceptors (oxidants) are employed, in whichthe lanes are as follows:

S. Protein Standard

Fib+2 mM Ru2+ only

Fibrinogen only

Fib+2 mM Ru2++NaPS

Fib+2 mM Ru2++Vit B12

Fib+2 mM Ru2++Cerium Sulphate

Fib+2 mM Ru2++Cerium Nitrate

Fib+2 mM Ru2++Oxalic acid

Fib+2 mM Ru2++Na-Periodate

Fib+2 mM Ru2++EDTA

FIG. 5 is a gel showing the results of cross-linking using alternativemetal-ligand complexes (catalysts), in which the lanes are as follows:

S. Protein Standard

Fibrinogen only

Fib+1 mM Ru2+ only

Fib+10 mM NaPS only

Fib+10 mM H₂O₂ only

Fib+1 mM Ru2++10 mM NaPS

Fib+1 mM Ru2++10 mM H₂O₂

Fib+1 mM Hemin only

Fib+1 mM Hemin+10 mM NaPS

Fib+1 mM Hemin+10 mM H₂O₂

FIG. 6 shows a photograph of an electrophoresis gel that demonstratesruthenium-catalysed photo-crosslinking of the additional matrix proteinsfibronectin and collagen.

Lanes: Gel A:

-   1. horse fibronectin-   2. horse fibronectin crosslinked with [Ru(bpy)₃]Cl₂-   5. Devro medical collagen (4 mg/ml); kangaroo tail-   6. Devro medical collagen (4 mg/ml); kangaroo tail, crosslinked with    [Ru(bpy)₃]Cl₂-   7. bovine fibrinogen-   8. bovine fibrinogen crosslinked with [Ru(bpy)₃]Cl₂

Gel B:

-   1. horse fibronectin-   2. horse fibronectin crosslinked with [Ru(bpy)₃]Cl₂-   3. Devro medical collagen (4 mg/ml); kangaroo tail-   4. Devro medical collagen (4 mg/ml); kangaroo tail, crosslinked with    [Ru(bpy)₃]Cl₂-   5. Chicken collagen-   6. Chicken collagen crosslinked with [Ru(bpy)₃]Cl₂-   7. bovine fibrinogen-   8. bovine fibrinogen crosslinked with [Ru(bpy)₃]Cl₂.

FIG. 7 demonstrates the rapid and efficient cross-linking of solublecollagen solutions using the photochemical process. Collagen in solution(Devro) was cross-linked using Ruthenium complex and white light for 30secs then run on 10% PAGE in denaturing conditions:

-   -   1. Kangaroo Tail Collagen 20 μg    -   2. Kangaroo Tail Collagen 20 μg Ruthenium cross-linked    -   3. Calf Skin Collagen 20 μg    -   4. Calf Skin Collagen 20 μg Ruthenium cross-linked revealing the        very high MW collagen polymer formed after 30 secs illumination        (lanes 2, 4)

FIG. 8 demonstrates the highly efficient cross-linking of fibronectinusing the photochemical process. Bovine fibronectin was purified frombovine plasma cryoprecipitate using gelatin-agarose chromatography,eluted with 3M urea. Shown are 4 different fibronectin-rich fractionsfrom the purification (lanes 2, 3, 4, 5) and the same followingcross-linking with Ru chemistry for 20 secs (lanes 6-9). Lanes 3 and 7shows that traces of fibrinogen present in that particular fraction alsoparticipates in the reaction.

FIG. 9 demonstrates the casting of a 3-D structure from fibrinogen,potentially serving as an implantable biocompatible prosthesis orscaffold. Fibrinogen solution (150 mg/ml) mixed with Ruthenium complexwas cast in a Lucite mould and illuminated for 30 secs with white light.Scale rule marked in mm.

FIG. 10 demonstrates a solid lens-shaped structure cast from collagensolution. A solution of bovine collagen (1%) was cast in a glasshemispherical well and cross-linked using Ru chemistry then dialysedagainst PBS for 24 hours.

FIG. 11 demonstrates using gel electrophoresis the highly rapid andefficient cross-linking of soluble fibrinogen. 4 mg/ml Pig Fibrinogen(Sigma) was cross-linked for 30 secs and run on Denaturing SDS-PAGE.Lanes 2, 4, 6: 10, 20, 50 microgram protein respectively; lanes 3, 5, 7same after cross-linking. Lanes 1, 8: MW standards. All subunits offibrinogen participate in the cross-linking reaction.

FIG. 12 demonstrates the cross-linking of soluble denatured bovine serumalbumin (BSA) Bovine serum albumin (BSA) at two concentrations (1 and 4mg/ml) was dissolved in two buffers (50 mM sodium Acetate pH 4.0 or 50mM tris-glycine, pH 9.0). Samples were heat denatured at 80 deg. for 60min. Gel shows various BSA samples (native and denatured; cross-linkedand uncross-linked; pH4.0 and pH9.0):

-   -   1. BSA 1 mg/ml Denatured pH 4.0 (1 hr, 80 deg C.)    -   2. BSA 1 mg/ml Denatured pH 4.0 Ruthenium cross-linked    -   3. BSA 4 mg/ml Denatured pH 4.0 Ruthenium cross-linked    -   4. BSA 1 mg/ml Native pH 4.0    -   5. BSA 1 mg/ml Native pH 4.0 Ruthenium cross-linked    -   6. BSA 4 mg/ml Native pH 4.0 Ruthenium cross-linked    -   7. BSA 1 mg/ml Denatured pH 9.0    -   8. BSA 1 mg/ml Denatured pH 9.0 Ruthenium cross-linked    -   9. BSA 4 mg/ml Denatured pH 9.0 Ruthenium cross-linked    -   10. BSA 1 mg/ml Native pH 9.0    -   11. BSA 4 mg/ml Native pH 9.0 Ruthenium cross-linked    -   12. Broad Range Standards

Native and denatured BSA are cross-linked at pH 4.0 (lanes 2, 3 and 5, 6respectively). Solid gels have also been prepared from 100 mg/ml BSA inbuffer at pH4.0. At pH 9.0 native and denatured BSA are onlyincompletely reactive showing the effect of pH on the proteininteraction.

FIG. 13 demonstrates that cells (chondrocytes) exposed to sodiumpersulphate for 60 mins retain viability within a range persulphatelevels. Human chondrocytic cells were pre-seeded onto Cultispher Sbeads. After growth for 6 days, an aliquot of cells on beads wereincubated in PBS containing the above concentrations of sodiumpersulphate for 1 hr then washed 3 times in PBS and stained with calceinAM for live cells (green) and ethidium homodimer for dead cells (red).

FIG. 14 demonstrates that cells (human chondrocytes) mixed with solubleporcine fibrinogen containing persulphate salt and Ru(Bpy)3 retainviability at 60 minutes before and after photo-activated cross-linking.Human chondrocytic cells were pre-seeded onto Cultispher S beads. Aftergrowth for 7 days, an aliquot of cells on beads, along with cells alone(without beads) were mixed with porcine fibrinogen (200 mg/ml), 10 mMammonium persulphate and 2 mM Ru(Bpy)3. Mixtures of cells on beads orcells alone in fibrinogen containing the photochemical reagents werecross-linked with blue light (5×20 s) and cell viability in uncured andcross-linked constructs were assessed at 60 minutes using calcein AM forlive cells (green) and ethidium homodimer for dead cells (red).

FIG. 15 demonstrates photo-activated cross-linking of gelatin intotissue culture scaffolds suitable for cell seeding. Various gelatintypes (A, B) from bovine and porcine origin with varying bloom strengthswere dissolved at 48° C. for 24 to 48 hrs, the pH adjusted to 7.0-7.5,and filter sterilised using a 0.45 μm filter. In this figure porcine 300bloom gelatin (—100 mg/ml) was mixed with 13.3 mM sodium persulphate and1.3 mM Ru(Bpy)3 and cross-linked with blue light (5×20 s). All solutionsformed firm plugs that remained solid upon heating to 56° C. (Day 0).Plugs were stable and remained sterile in PBS at 37° C. up to 9 days.

FIG. 16 demonstrates biocompatibility of photo-activated cross-linkedgelatin plugs with mouse NR6 fibroblasts. Sterile gelatin plugs,prepared as described in FIG. 9, were seeded with 60×10³ NR6 mousefibroblasts in 1.2 ml DMEM culture medium containing 10% FBS in 24 welltissue culture plates, and incubated for 5 days at 37° C. Cells remainedviable over the culture period with no signs of toxic, leachablecompounds from the photochemical process. Cell viability was assessedusing calcein AM for live cells (green) and ethidium homodimer for deadcells (red).

FIG. 17 demonstrates viability of cells (human chondrocytes) after 24hrs entrapped within various photo-activated cross-linked gelatins.Human chondrocytic cells were pre-seeded onto Cultispher S beads. Aftergrowth for days, an aliquot of cells on beads was mixed with gelatinfrom porcine and bovine origin with different bloom strengths as shown.Photo-activated agents were added and gelatin solutions with cells onbeads were cross-linked. After 24 hrs, cell viability was assessed usingcalcein AM for live cells (green) and ethidium homodimer for dead cells(red). Top row shows low power images of viable cell/beads distributionwith the cured gelatin plugs. Bottom rows show higher power confocalimages visualising live cells attached on beads within a dark gelatinmatrix background.

FIG. 18 demonstrates cell migration within the photo-activatedcross-linked gelatin matrix. Human chondrocytes were prepared, mixedwith porcine gelatin and cross-linked as in FIG. 17. Cell migration wasassessed day 1 to day using normal transmission microscopy as well asusing fluorescence staining of viable cells as indicated in FIG. 11.

FIG. 19 shows graphically the mechanical properties of photocross-linked fibrinogen hydrogel. A solution of fibrinogen (150 mg/ml)was prepared in phosphate-buffered saline and ruthenium trisbipyridyland sodium persulphate were added at 2 mM and 20 mM finalconcentrations, respectively. A dumbbell-shaped strip (30 mm×4 mm×1 mm)with stainless-steel strips at each end was mounted in the tensiletester and extension increased by steps of 20% strain until the stripfailed.

FIG. 20 shows graphically data generated using a Photorheometer with atunable light source and a 400 nm-500 nm filter. The data shows theshear modulus (G^(˜)) before and after turning on the light (at 1 min)measuring a solution containing 150 mg/ml fibrinogen, 2 mM Ru(II)(bpy)₃²⁺ and 20 mM sodium persulfate in PBS. The reaction is complete within 1min. Light intensity was 21 mW/cm². Shear modulus reaches 50 KPa.Duplicate plots overlayed; 3 data points/sec were sampled.

FIG. 21 shows the result of treating two concentrations of fibrinogenfor 2 minutes at room temperature with thrombin. Panel A shows a clotformed from a 5 mg/ml solution of fibrinogen (similar to theconcentration of fibrinogen in blood—ref: Weisel J W. Fibrinogen andfibrin. Adv Protein Chem. 2005; 70:247-99). Panel B shows a stiffer clotformed from a 50 mg/ml solution of fibrinogen. Both fibrinogen solutionswere treated with 10.5 U of thrombin at room temperature. Both clotswere completely soluble in 2.5% acetic acid within 2 minutes at roomtemperature. Panel C shows photochemically crosslinked fibrin (samplestreated as in A, but 2 mM ruthenium tris-bipyridyl and 20 mM sodiumpersulphate added simultaneously with thrombin in the dark). The sampleswere then illuminated with white light (600 W tungsten halide lamp) for10 seconds. Samples were subsequently soaked in 2.5% acetic acid (“5” isfibrinogen at 5 mg/ml; “50” is fibrinogen at 50 mg/ml) and wereinsoluble as shown. Panel D shows a fibrinogen sample 5 mg/ml) treatedwith 2 mM ruthenium tris-bipyridyl and 20 mM sodium persulphate, addedsimultaneously with thrombin in the dark. The fibrin clot wassubsequently transferred in the dark to a solution of 2.5% acetic acid.After 2 minutes at room temperature, the clot dissolved completely,demonstrating that, without illumination, no covalent crosslinkingoccurred in the fibrin clot.

FIG. 22 is a photograph showing foamed, photo-crosslinked fibrinogenscaffolds, seeded with C2C12 cells and implanted subcutaneously intonude mice. Scaffolds and surrounding tissue were sectioned and stainedwith Masson's trichrome at 2 weeks (A) and 4 weeks (B) afterimplantation. Arrows indicate multinucleated myotubes and blood vessels.

DETAILED DESCRIPTION OF THE INVENTION

In one form the invention relates to the preparation of manufacturedarticles. In an embodiment there is provided a method of manufacturingan article, comprising the steps of:

(1) providing a preferentially associating protein solution, aphotoactivatable metal-ligand complex and an electron acceptor;

(2) irradiating the protein solution to photoactivate thephotoactivatable metal-ligand complex, thereby initiating across-linking reaction to form a 3-dimensional matrix of the protein.

In an embodiment the manufactured article is selected from mouldedarticles such as dressings and pads, implants, lens, tubes, beads andfibres, sponges and sheets.

In an embodiment the manufactured article is a scaffold for tissueengineering or cell-based therapies.

Alternatively the manufactured article maybe a scaffold for use in nonmedical applications such as cell culture or water retention beads.

Thus an embodiment relates to a method of preparing a scaffold fortissue engineering or cell-based therapies, comprising the steps of:

(1) providing a preferentially associating protein solution, aphotoactivatable metal-ligand complex and an electron acceptor;

(2) irradiating the protein solution to photoactivate thephotoactivatable metal-ligand complex, thereby initiating across-linking reaction to form a 3-dimensional matrix of the protein.

In an embodiment the protein solution is introduced to a shaped vesselcapable of transmitting light so as to allow shaped articles to beproduced. Alternatively the solution may be irradiated without a guideto shape the article, and the product will be formed as fibres or beads.Additionally the solution may be sprayed or printed.

In an embodiment the irradiation is conducted prior to implantation ofthe scaffold or prior to use of the scaffold. However the irradiationmay be carried out in full or in part following introduction of theprotein solution, be that following partial cross-linking or otherwise,to a patient. Advantageously the protein solution is partiallycross-linked to facilitate shaping, and typically is in the form of ahydrogel when introduced to the patient. In an embodiment the hydrogelis injectable.

In a further embodiment there is provided a manufactured articlecomprising a 3-dimensional matrix of a protein which is capable ofpreferential association through the interaction of hydrophobic aminoacid side chains, wherein said 3-dimensional matrix comprises saidprotein cross linked by covalent bonds formed between amino acid sidechains juxtaposed through the interaction of hydrophobic amino acid sidechains of the protein.

In a still further embodiment there is provided a scaffold for tissueengineering comprising a 3-dimensional matrix of a protein which iscapable of preferential association through the interaction ofhydrophobic amino acid side chains, wherein said 3-dimensional matrixcomprises said protein cross linked by covalent bonds formed betweenamino acid side chains juxtaposed through the interaction of hydrophobicamino acid side chains of the protein.

In an embodiment the protein is a matrix protein. In particular theprotein may be selected from, but not limited to the group consisting offibrinogen, fibrin, collagen, keratin, gelatin, fibronectin, andlaminin, or admixtures thereof.

In an embodiment the protein is a globular protein in which preferentialassociation has been induced by chemical modification or unfolding.Unfolding of a protein may be induced by raising or lowering the pH,decreasing or increasing the ionic strength of a protein solution or inother ways know to the person skilled in the art. For example, at pH 4.0serum albumin is transformed from the “normal” (N) configuration to the“fast (F) configuration. Chemical modification may include addition ofattached residues such as tyrosine residues under mild conditions withBolton-Hunter reagent.

In an embodiment the article is in the form of a hydrogel and such ahydrogel is particularly useful for applications such contact lens,breast implants, reservoirs for drug delivery systems, protective layerson stents and implants so as to provide, for example, absorption,desloughing and debriding capacities of necrotics and fibrotic tissueand for use as a scaffold for tissue engineering or in cell delivery.The article could also be useful in agricultural applications such as aslow release fertilising bead.

In an embodiment the vessel is a transparent mould adapted to produce ashaped article. This may be of tubular design, for example forintroduction as part of a lumen of a blood vessel, duct or tract such asthe urinary tract. Alternatively the shaped article may be a prosthesisshaped to fit a particular surface, a sheet or mat, a membrane or asponge.

In an embodiment the protein is introduced into a tissue or tissuedefect and irradiated in situ to photoactivate the photoactivatablemetal-ligand complex. The product may be introduced directly withoutconcern for the shape. For example, a protein solution may be preparedwith live cells as inclusions and cured in situ before, during or afterinjection.

In an embodiment the protein solution further comprises a therapeuticagent selected from the group consisting of cells, growth factors,bioactive agents and nutrients. In an embodiment a drug (particularly achemotactic, growth promoting or differentiation factor but also aconventional drug such as an antibiotic or chemotherapeutic drug) isintroduced to the protein solution. While not wishing to be bound bytheory it is believed that the therapeutic agent is captured in the3-dimensional matrix formed by the cross-linking reaction and soretained in situ for an extended period before the matrix degrades.

Additionally, the present inventors have found that the photochemicalreaction described herein can create covalent cross-links betweenendogenous proteins if applied to a tissue. Thus in an embodiment thereis envisaged a method of joining and/or sealing tissues in a surgicalprocedure or medical treatment, comprising the steps of:

(1) applying a photoactivatable metal-ligand complex and an electronacceptor to a tissue portion;

(2) irradiating said tissue portion to photoactivate thephotoactivatable metal-ligand complex;

thereby initiating a cross-linking reaction between one or moreendogenous matrix proteins to seal said tissue portion or join saidtissue portion to an adjacent tissue portion.

In particular, the photochemical reaction described herein can formcovalent crosslinks in a fibrin matrix such as that formed when thrombincatalyses the conversion of fibrinogen into fibrin in haemostasis. Thusit may be used to enhance the adhesive strength of the clot formed whenhaemostasis takes place in vivo.

In an embodiment thrombin is applied to the tissue whereby fibrin isformed by cleavage of endogenous fibrinogen under the influence of theapplied thrombin and said fibrin is involved in the photoactivatedcross-linking reaction. It will also be appreciated that a combinationof prothrombin and calcium applied to a wound site can also serve as asource of thrombin.

The method may involve moving the said tissue portion to a positionadjacent, inclusive of touching, the adjacent tissue portion, wherenecessary, such as when a relatively large gap exists between them.Alternatively, the matrix resulting from the cross-linking reaction mayform a plug which nevertheless binds the tissues to either side of anincision or opening. Furthermore, the cross-linked matrix may form acoating over a region of tissue, and may be shaped or supported asappropriate, for example, the thrombin and/or photoactivatablemetal-ligand complex and electron acceptor may be carried by a collagensheet or impregnated in a surgical gauze or fleece. Accordingly, it willbe appreciated that the cross-linked matrix can adopt a physical form tosuit the application in which it is used, and it will be applied in theappropriate manner to suit that purpose.

In an embodiment the method is used to seal a vessel. This may be toseal blood vessels to prevent blood loss, to treat lung tissue forsealing air leaks, to prevent cerebrospinal fluid leakages or to seal avessel to prevent leakage of any other biological fluid.

In an embodiment the method is used to join a first tissue portion and asecond tissue portion to seal a wound such as an incision, for example,in aesthetic or cranial surgery.

In an embodiment the method is used to treat a soft tissue such as liveror lung tissue which has suffered injury, for example, by coating thetissues.

Tissue adhesives of the present invention may also be used as wounddressings, for example, if applied alone or in combination with adhesivebandages, or as a haemostatic dressing in the operating room.

In an embodiment there is provided a closure for a leaking woundcomprising a substrate suitable for application to a wound to stemleakage, wherein said substrate is impregnated or coated with aphotoactivatable metal-ligand complex and an electron acceptor.

In an embodiment the closure further comprises thrombin.

In an embodiment the substrate is a bandage, gauze, cloth, tampon,membrane or sponge.

In this embodiment there is also envisaged a method of stemming bleedingfrom a wound comprising applying a closure as described to the wound andirradiating the closure and surrounding tissue, thereby causing across-linking reaction between fibrin formed by cleavage of endogenousfibrinogen under the influence of the thrombin within or coating theclosure and one or more endogenous matrix proteins in the surroundingtissue to join the closure to the surrounding tissue.

According to a further aspect of the present invention there is provideda kit comprising a thrombin, a photoactivatable metal-ligand complex andan electron acceptor.

In an embodiment the thrombin, metal-ligand complex and an electronacceptor are separately contained within the kit.

The kit optionally contains buffer, such as phosphate buffered saline,for preparation of solutions of one or more thrombin, photoactivablemetal-ligand complex and electron acceptor. The kit may include a weakacid such as acetic acid to render an otherwise insoluble matrix proteinsuch as fibrin soluble.

A light source may also be provided in the kit, particularly where thekit is for use in the field.

In an embodiment a wound closure such as a bandage, gauze, cloth,tampon, membrane or sponge may be provided in the kit and, optionally,maybe pre-impregnated or pre-coated with thrombin, a photoactivablemetal-ligand complex and an electron acceptor.

In an embodiment, a composition comprises one or more of thrombin, aphotoactivatable metal-ligand complex and an electron acceptor and inertcarrier. In particular, these compounds are dissolved in an inertcarrier, and a solution comprising all three components is applied tothe tissue portion. In particular, the solution is an aqueous solution,and generally a solution in a buffer such as phosphate buffered saline.Alternatively, each of the three components could be applied separately,or as separate solutions, prior to irradiation.

The method of application is not critical and may involve spreading of asolution over the appropriate tissues or over a region to be sealed orrubbing of one tissue portion on another to spread the solution.

In an embodiment the thrombin composition further comprises human bloodfactor XIII. Factor XIII is a compound that strengthens blood clots byforming covalent cross-links between strands of fibrin.

In an embodiment the thrombin composition further comprises aprotininand factor XIII. Aprotinin is a protein that inhibits the enzymes thatbreak down blood clots.

In an embodiment a drug (particularly a chemotactic, growth promoting ordifferentiation factor but also a conventional drug such as anantibiotic or chemotherapeutic drug) or other therapeutic agent isapplied to said first tissue portion and/or said second tissue portion,in particular, as a component of the composition described above. Whilenot wishing to be bound by theory it is believed that the therapeuticagent is captured in the matrix formed by the cross-linking reaction andso retained in situ for an extended period before the matrix degrades.

In another embodiment, there is envisaged a method of joining and/orsealing at least one substrate, comprising the steps of:

(1) applying a protein or peptide solution, a photoactivatablemetal-ligand complex and an electron acceptor to at least one substrate;

(2) irradiating said material to photoactivate the photoactivatablemetal-ligand complex;

thereby initiating a cross-linking reaction to adhere or join saidsubstrate to an adjacent substrate.

In one embodiment the protein may be a partially denatured protein suchas serum albumin or gelatin or alternatively a matrix protein such asfibrinogen or collagen.

In an embodiment the protein is applied to a surface of one or moreitems to be joined or sealed and irradiated in situ to photoactivate thephotoactivatable metal-ligand complex to adhere one item to another orseal the surface of one or more items. Thus the protein may be used asan adhesive or a sealant and photoactivated to form the adhesive link ormake the seal. The person skilled in the art will appreciate thatnumerous items may be joined or sealed in this way including itemsmanufactured from glass, metal, wood, thermoplastic or thermosettingpolymeric materials, and so on. The use as an adhesive or sealant mayinvolve the connection or sealing of items without restriction as toform, and includes household items, timber products and manufacturedgoods. Thus it is envisaged that the sealant may be used as an adhesivein non medical applications such as in joining wood products in mendingunderwater pipes, in labelling of bottles. These crosslinked materialsprovide a non toxic, strong alternative adhesive to the currently usedadhesives.

The present inventors have made the unexpected observation thatself-association can be induced and/or enhanced via total or partialdenaturation of a peptide or protein or by chemical modification whichrenders proteins previously in the native state susceptible tophotochemical cross linking.

In an embodiment there is provided a method of joining and/or sealingmaterials in a surgical procedure, medical treatment, comprising thesteps of:

(1) applying an at least partially denatured protein, a photoactivatablemetal-ligand complex and an electron acceptor to a substrate;

(2) irradiating said substrate to photoactivate the photoactivatablemetal-ligand complex;

thereby initiating a cross-linking reaction to form a matrix comprisingsaid at least partially denatured protein to seal or join said substrateto an adjacent substrate;

wherein said at least partially denatured protein has been rendered moresusceptible to cross-linking compared to its native state.

In a preferred embodiment said substrate is a tissue portion.

In an embodiment said at least partially denatured protein is an atleast partially denatured matrix protein and said matrix protein isselected from the group consisting of fibrinogen, fibrin, collagen,fibronectin, keratin and laminin, or admixtures thereof.

In an embodiment denaturation occurs by heating.

In an alternative embodiment said at least partially denatured proteinis a globular protein in which preferential association has been inducedby chemical modification or unfolding. Unfolding of a protein may beinduced by raising or lowering the pH, decreasing or increasing theionic strength of the solution, or in other ways, known to a personskilled in the art.

In an embodiment physical or mechanical denaturation techniques areemployed. Typically a mechanical force such as stirring or agitation ofa protein solution is used to produce a “foam”. This may find particularapplication where a space filling sealant or adhesive is desirable.Alternatively compressed gas could be introduced to the protein solutionto achieve denaturation and to facilitate delivery of the sealant.

In an embodiment denaturation occurs by way of acid/alkali treatment orpH adjustment.

In an embodiment said at least partially denatured protein is gelatin.Gelatin is produced by the partial hydrolysis of collagen, which causesthe natural molecular bonds between individual collagen strands to bebroken down into a denatured form. Typically hydrolysis occurs bylowering the pH.

In an embodiment said at least partially denatured protein is serumalbumin in which the “fast” (F) configuration has been induced.Typically the F configuration is induced by reduction of the pH to 4.0and occurs through dissolution of serum albumin in a weakly acidicsolution or by addition of a weak acid to an aqueous solution. The N toF transition involves the unfolding of domain III, with an increase inviscosity, much lower solubility and a decreased helical content. Whilenot wishing to be bound by theory, it is believed that this transitionreveals hydrophobic residues which would otherwise have been locatedinternally in this (in the N configuration) globular protein and soserum albumin self associates when in the F configuration at pH 4.0.

In an embodiment chemical modification is employed to render susceptiblethe protein to cross-linking compared to its native state. Such chemicalmodification may include the modification of amino acid side chains toinclude of aromatic moieties such as the phenolic moiety present intyrosine. By way of example primary amines such as the lysine residuesin a protein may be modified under mild conditions with Bolton-Hunterreagent (N-succinimidyl-3-[4-hydroxyphenyl]propionate) or water solubleBolton-Hunter reagent (sulfosuccinimidyl-3-[4-hydroxyphenyl]propionate).Equally it may involve modification of the protein to alter itssecondary, tertiary or quaternary structure. Protein modification agentsare well known to the person skilled in the art and include reagentswhich can effect sulfhydryl reduction, addition of sulfhydryl or aminogroups, protein acylation, etc.

Tissue adhesives of the present invention may also be used as wounddressings, for example, if applied alone or in combination with adhesivebandages, or as a hemostatic dressing in the operating room. Adhesivesof the present invention may also be used in non-medical applicationssuch as in pipe repair, labelling of bottles, as well as in generaladhesive and sealing applications.

Accordingly, an embodiment provides a closure for a bleeding woundcomprising a substrate suitable for application to a wound to stembleeding, wherein said substrate is impregnated or coated with an atleast partially denatured protein, a photoactivatable metal-ligandcomplex and an electron acceptor, wherein said at least partiallydenatured protein or chemically modified protein is rendered moresusceptible to photochemical cross-linking compared to its native state.

In an embodiment the substrate is a bandage, gauze, cloth, tampon,membrane or sponge.

Additionally this embodiment provides a method of stemming bleeding froma wound comprising applying a closure as described to the wound andirradiating the closure and surrounding tissue.

In a further embodiment there is provided a composition comprising an atleast partially denatured protein, a photoactivatable metal-ligandcomplex and an electron acceptor, wherein said at least partiallydenatured protein or chemically modified protein is rendered susceptibleto photochemical cross-linking compared to its native state orsusceptibility to photochemical cross-linking is enhanced.

A further embodiment provides a kit comprising an at least partiallydenatured protein, a photoactivatable metal-ligand complex and anelectron acceptor, wherein said at least partially denatured protein orchemically modified protein is rendered more susceptible tophotochemical cross-linking compared to its native state.

In an embodiment the at least partially denatured protein, metal-ligandcomplex and an electron acceptor are separately contained within thekit.

The kit optionally contains buffer, such as phosphate buffered saline,for preparation of solutions of one or more of the denatured protein,photoactivable metal-ligand complex and electron acceptor. The kit mayinclude a weak acid such as acetic acid to allow for in situdenaturation.

A light source may also be provided in the kit, particularly where thekit is for use in the field.

In an embodiment a wound closure such as a bandage, gauze, cloth,tampon, membrane or sponge may be provided in the kit and, optionally,maybe pre-impregnated or pre-coated with an at least partially denaturedprotein, a photoactivable metal-ligand complex and an electron acceptor.

In an embodiment, a composition comprises one or more of an at leastpartially denatured protein, a photoactivatable metal-ligand complex andan electron acceptor and inert carrier. In particular, these compoundsare dissolved in an inert carrier, and a solution comprising all threecomponents is applied to the tissue portion. In particular, the solutionis an aqueous solution, and generally a solution in a buffer such asphosphate buffered saline. Alternatively, each of the three componentscould be applied separately, or as separate solutions, prior toirradiation.

In an embodiment a drug (particularly a chemotactic, growth promoting ordifferentiation factor but also a conventional drug such as anantibiotic or chemotherapeutic drug) or other therapeutic agent isapplied to said first tissue portion and/or said second tissue portion,in particular, as a component of the composition described above. Whilenot wishing to be bound by theory it is believed that the therapeuticagent is captured in the matrix formed by the cross-linking reaction andso retained in situ for an extended period before the matrix degrades.

Modes for Performing the Invention

At least in preferred embodiments the invention provides a rapid,specific and biocompatible process for covalent cross-linking ofselected proteins (soluble and/or insoluble), including ECM proteins.The articles which result may be used in contact lens, sheet dressingsof wound dressings or pads, as breast implants, as cartilage implants,as a component in nerve sheaths and novel blood vessel constructs, ascomponents in water retention applications or 3 dimensional cell culturematrices or in orthopaedic applications.

The materials are porous and potentially resorbable and so particularlyuseful as scaffolds that can be used directly as injectable hydrogels orengineered into structures for e.g. delivery of cells; reconstruction ofsoft tissues/organs including skin; directed migration and population byendogenous cells; delivery or augmentation of enzymes/growth factorsetc.; also cross-linking or treatment of acellular or naturally derivedtissues/organs; also scaffolds for culturing cells. Additionally controlcan be exerted over the biomechanical properties of the materials byvarious means e.g. composition of the protein matrices and regulation ofdegree of cross-linking. Advantageously, bonding of thematrices/scaffolds to endogenous ECM at the site of application isbelieved to take place. Furthermore, degradation/resorption rates can bevaried by controlling the parameters outlined above.

The present inventors have found a means of preparing tissue scaffoldsand matrices using soluble ECM components or other molecules whichpreferentially associate and form covalently linked, high-molecularweight hydrogels using a novel, cell-compatible process. The process canbe used to construct stable, proteinaceous scaffolds for the delivery ofcells or for organ and tissue reconstruction as well as matricessuitable e.g. for burns and open wound treatment, soft tissue implantsand cell culturing.

The cross linking imparts a mechanical stability and degradation controlthat is lacking in the current materials. Another major advantage ofthis system is the controllable and rapid gelation of preferentiallyassociating protein or peptide solutions, which allows in-situ curing tobe carried out. Injectable gels as well as prostheses can be formulatedand cured to form a biocompatible, covalently cross-linked network withmechanical properties matched to the surrounding tissue or ECM. Theporous structure and protein composition of cross-linked polymermatrices promotes tissue regrowth and the rate of degradation can betailored to complement the rate of tissue repair. Because thecross-linking process is essentially biocompatible the system can beused as a delivery vehicle for cells, growth factors, bioactive agentsand nutrients. Thus the physical, mechanical and biologicalcharacteristics can be tailored to specific needs both in medical andnon medical applications.

The present inventors have recognised that the natural strongpreferential association of peptides or proteins would likely result inthe inter- and intra-molecular conjunction of a number of individualaromatic amino acid residues such as tyrosine and histidine, mostparticularly, tyrosine residues. They have inferred that this wouldrender preferentially associated proteins susceptible to covalentbonding and polymerisation using a photoactivatable catalyst capable ofinducing formation of a stabilised free radical on adjacent side-chainsso as to initiate formation of a carbon-bond between the two.Consequently they have successfully cross-linked proteins in aphoto-initiated chemical process in which a metal-ligand complex inconjunction with an electron acceptor directly mediates cross-linkingbetween adjacent proteins through a mechanism which does not involveformation of potentially detrimental species such as singlet oxygen,superoxide or hydroxyl radicals. While not wishing to be bound bytheory, it is believed that the mechanism involves irradiation of themetal-ligand complex to induce an excited state, followed by transfer ofan electron from the metal to an electron acceptor. The oxidised metalthen extracts an electron from a side chain such as a tyrosine sidechain in the protein to produce, a tyrosyl radical which reactsimmediately with a nearby tyrosine to form a dityrosine bond. A directcross-link (without any bridging moiety) is created quickly in thisphoto-initiated chemical reaction, without the need for introduction ofa primer layer and without the generation of potentially detrimentalspecies such as singlet oxygen, superoxide and hydroxyl radicals.

The term “photoactivatable metal-ligand complex” as used herein means ametal-ligand complex in which the metal can enter an excited state whenirradiated such that it can donate an electron to an electron acceptorin order to move to a higher oxidation state and thereafter extract anelectron from a side chain of an amino acid residue of a matrix proteinto produce a free radical without reliance upon the formation of singletoxygen. Suitable metals include but are not limited to Ru(II), Pd(II),Cu (II), Ni (II), Mn (II) and Fe (III) in the form of a complex whichcan absorb light in the visible region, for example, an Ru(II) bipyridylcomplex, a Pd(II) porphyrin complex, a sulfonatophenyl Mn (II) complexor a Fe(III) protoporphyrin complex, more particularly, an Ru(II)bispyridyl complex or a Pd(II) porphyrin, in particular, an Ru(II)(bpy)₃complex such as [Ru(II) (bpy)₃]Cl₂. Efficient cross-linking occurs inthe presence of an electron acceptor, and requires only moderateintensity visible light. The options and types of chemistry involved areoutlined in Brown et al (2001) the contents of which are incorporatedherein by reference.

As used herein the term “electron acceptor” refers to a chemical entitythat accepts electron transferred to it and so refers to an easilyreduced molecule (or oxidizing agent) with a redox potentialsufficiently positive to facilitate the cross-linking reaction. A rangeof electron acceptors will be suitable. In an embodiment the electronacceptor the electron acceptor is a peracid, a cobalt complex, a cerium(IV) complex or an organic acid. Typically the electron acceptor is apersulfate, periodate, perbromate or perchlorate compound, vitamin B12,Co (III) (NH₃)₅Cl²⁺, cerium(IV) sulphate dehydrate, ammonium cerium(IV)nitrate, oxalic acid or EDTA. Preferably the persulfate anion is used asthe electron acceptor, as it is one of the strongest oxidants available.The standard oxidation reduction potential for the reaction

S₂O₈ ²⁻+2H⁺+2e⁻=>2 HSO₄ ⁻

is 2.1 V, as compared to 1.8 V for hydrogen peroxide (H₂O₂). Thispotential is higher than the redox potential for the permanganate anion(MnO₄ ⁻) at 1.7 V, but slightly lower than that of ozone at 2.2 V.

As used herein the term “matrix protein” refers to isolated and purifiedforms of the proteins which are abundant and common in the extracellularmatrix of animals. Typical matrix proteins are fibrinogen, fibrin,collagen, fibronectin, keratin, laminin, elastin; or admixtures thereof,and these may be isolated from human or animal sources or prepared, forexample, using recombinant DNA technology. As well, the inventors haveobserved that the preferentially associating proteinsbeta-lactoglobulin; gelatin; glycinin; glutens; gliadins and resilin canbe rendered into hydrogels using the process described herein, and mayfind application in particular embodiments of the invention. Inaddition, derivatives of these compounds, including peptide derivativesor extracts containing them are suitable for use in the presentinvention, and they may be used in admixture. Furthermore they may bepresent as native proteins or may be denatured and, providedpreferential association will take place, present under a range ofconditions such as high or low pH, high or low salt concentrations andin aqueous or non-aqueous solution.

As herein the term “protein solution” refers to a solution or dispersionof one or more peptides or proteins in a solvent or a solvent mixture.Typically the solution is an aqueous solution and may include aco-solvent such as ethanol, and may be a buffer solution. The termincludes a dispersion of hydrated protein or denatured protein granules.The protein solution may comprise a single peptide or protein or amixture of peptides or proteins having the property of self association,whether these be peptides or proteins that preferentially associate intheir natural state or not, as peptides or proteins which possess theinherent property of self association but do not occur together or donot preferentially associate in nature may nevertheless associate whenin admixture in solution.

As used herein the term “aromatic amino acid” refers to an α-amino acidin which the side chain comprises a substituted or unsubstituted aryl orheteroaryl group. The 20 or so common, naturally-occurring amino acidsinclude the aromatic amino acids phenylalanine, tyrosine and tryptophanand histidine.

As used herein the terms “fibrin” and “fibrinogen” encompass fibrin andfibrinogen themselves, purified fibrin or fibrinogen sub-units orcomposites or admixtures thereof. These might be isolated from human oranimal whole blood or plasma. Alternatively these products or activehomologs or fragments thereof may be prepared by genetic engineering,and such products are also envisaged for use in the present invention.For example, Pharming is developing three fibrinogen genes (rTS) underthe transcriptional control of the bovine α-S1 casein promoter toachieve high level, mammary gland-specific expression. Nuclear transfertechnology has been used to generate a number of transgenic cows thatshow expression levels of human fibrinogen in the milk at levels of 1-3g/l.

The inventors have also demonstrated that clotted fibrin itself(produced by treatment of soluble fibrinogen with thrombin and insolublein phosphate buffer) can be rendered soluble by, e.g. addition of 2%acetic acid or other means, and this can also subsequently becross-linked using the method of the invention.

As used herein the term “soluble fibrin” refers to fibrin that has beenprepared from fibrinogen by, for example, hydrolysis with thrombin, thenrendered soluble by addition of a weak acid such as 2% acetic acid, achemical chaotrope such as urea, or other means.

As used herein the term “applying” or “apply” or “application” refers tosequential application of the matrix protein, photoactivatablemetal-ligand complex and the electron acceptor in any order or toapplication of compositions comprising any one or more of the matrixprotein, photoactivatable metal-ligand complex and the electronacceptor. The matrix protein, photoactivatable metal-ligand complex andthe electron acceptor or compositions containing them in admixture maybe provided in solid form such as a lyophilized powder or a plug ofmaterial or in liquid such as a solution or foam.

As used herein the term “self associate” or its equivalents “selfassociates”, “self associating”, “self association” and the like, referto the inherent property of a protein to associate with itself throughhydrophobic interaction or bonding i.e. through the association ofnon-polar groups or domains in aqueous media due to the tendency ofwater molecules to exclude non-polar species. Salt bridges can alsocommonly occur to facilitate and stabilize protein and peptideinteractions. The matrix proteins typically self associate in theirnatural configuration in aqueous media. It will be appreciatedadditionally that self association can be induced by altering thenatural configuration, for example by inducing some unfolding of aprotein such as by altering the pH or ionic strength of the media. Forexample, at pH 4.0 bovine serum albumin is transformed from its normal(N) configuration to the so-called “fast” (F) configuration, and the Nto F transition involves the unfolding of domain III, with an increasein viscosity, much lower solubility and a decreased helical content.While not wishing to be bound by theory, it is believed that thistransition reveals hydrophobic residues which would otherwise have beenlocated internally in this (in the N configuration) globular protein andso it self associates when in the F configuration at pH 4.0.

As herein the term “preferentially associate” “preferentially associate”or its equivalents “preferentially associates”, “preferentiallyassociating”, “preferentially association” and the like refers topeptides and proteins that have the inherent characteristic ofself-association but which, if brought into juxtaposition with anotherprotein having this characteristic will associate with it. Therefore aself associating protein is, in effect, a preferentially associatingprotein. Additionally, the term refers to peptides and proteins thathave specific interaction domains through which they interact andassociate through hydrophobic interaction or bonding i.e. through theassociation of non-polar groups or domains in aqueous media due to thetendency of water molecules to exclude non-polar species. Salt bridgescan also commonly occur to facilitate and stabilize protein and peptideinteractions. By way of example collagen and fibronectin and fibrinogenand fibronectin possess domains which preferentially bind to each otherso that naturally self associate.

As used herein the term “tissue” refers to a plurality of cells locatedin close juxtaposition, be they alike in character or unlike, and soincludes a tissue in the histological sense such as muscle tissue butalso includes discrete structures such as the walls of a vessel like ablood vessel and the surface of an organ, including a raw, cut surface.The usage of the term should be read in conjunction with the intendeduses described herein, and is not intended to limit the uses described.

As used herein the term biomaterial refers to an article that has beenmade from proteinous or peptide material and that is preferentially formedical use, such as for use as an implant, as a tissue sealant or as atissure scaffold. In the alternative the biomaterial could be used fornon-medical use as described herein. It will be appreciated that thebiocompatibility of such materials is not necessarily advantageous, asit is in medical use, but non-medical applications are not excluded forthis reason solely.

Composite materials may be used to regulate the bulk properties of thebiomaterial (stiffness, elasticity or modulus) so that the hydrogel thusformed has bulking or filling properties suitable for tissue implants orprostheses that may be naturally bonded to surrounding tissues. Suitablematerials might include mineral, metal or inorganic inclusions (e.g.hydroxyapatite or nanocrystalline titanium or other metal salts),synthetic organic compounds (plastics or other polymers) or naturalorganic polymers (e.g. chitin, chitosan or cellulosic materials).

In an embodiment the protein is present in a solution or solutionmixture, typically a solution with a protein concentration in the rangeof 0.1-20% w/v, preferably 0.5-10% and most often 0.5-2% or more forcollagen; typically 5% or more for other proteins, e.g. fibrinogen. Theperson skilled in the art will appreciate that solutions with a higherconcentration of protein may be effectively cross-linked but economicconsiderations dictate that very high concentrations of protein will notbe used, and that there is a limit to the concentration of protein whichwill remain in solution. Likewise, solutions with a lesser concentrationof protein may be cross-linked although the gel resulting from thisprocedure may be less effective.

In an embodiment an appropriate concentration of single protein solution(typically 0.5-2% or more for collagen; typically 5% or more for otherproteins, e.g. fibrinogen) or composites of suitable proteins insolvent, solvent mixture or buffer is mixed with 2 mM Ru(Bpy)3 and 20 mMpersulphate salt (sodium, ammonium, potassium etc.) and irradiated withwhite light (450 nm nominal wavelength) for at least 10 secs. to formthe hydrogel. This process is cell compatible. To form 3-dimensionalstructures the biomaterial can be cast or contained within transparentmoulds. If performed in situ the process will covalently link thehydrogel thus formed with surrounding tissues with a natural affinity(e.g. those containing integrins) thereby forming an endogenous bond.

To determine the effect of cross-links and the optimal number ofcross-links per monomer unit, the resilience of a cross-linked proteincan be measured using methods known in the art. The level ofcross-linking can vary. For example, the degree of cross-linking is afunction of the time and energy of the irradiation. The time required toachieve a desired level of cross-linking may readily be computed byexposing non-cross-linked polymer to the source of radiation fordifferent time intervals and judging the suitability of the resultingcross-linked material for each time interval. By this experimentation,it will be possible to determine the irradiation time required toproduce an appropriate material for a particular application (see, e.g.,U.S. Pat. No. 4,474,851, the contents of which are incorporated hereinby reference).

The ability to tune the cross-linking by changing the irradiationconditions renders the resultant biomaterial, manufactured article,sealant or adhesive highly versatile.

The proteins are preferably lightly cross-linked. Preferably, the extentof cross-linking is at least about one cross-link for every five or tento one hundred monomer units, e.g., one cross-link for every twenty tofifty monomer units. The extent of cross-linking may be monitored duringthe reaction or pre-determined by using a measured amount of reactants.For example, since the dityrosine cross-link is fluorescent, thefluorescence spectrum of the reactant mixture may be monitored duringthe course of a reaction to determine the extent of cross-linking at anyparticular time. This allows for control of the reaction and theproperties of the scaffold which results.

The photochemical reaction described above can form covalent crosslinksin a extracellular protein matrix. The inference is that addition of aphotoactivatable metal-ligand complex and an electron acceptor to awound where endogenous extracellular matrix proteins are present orwhere thrombin is present and induction of a photochemical process, willinduce or enhance the clot formation as a haemostatic agent in vivo.While not wishing to be bound by theory, it is believed that theruthenium complex-mediated protein oxidation carries out a very rapidcovalent crosslinking reaction, thereby stabilising the thrombin-derivedclot in a manner analogous to the function of Factor XIII-mediatedcrosslinking (Lee M G and Jones D (2005)).

Therefore in embodiments of the invention addition of a photoactivatorsuch as ruthenium tris-bipyridyl chloride and an electron acceptor suchas sodium persulphate (or an equivalent salt) to a solution of thrombin,followed by treatment of, e.g. a tissue or wound site with thecomposition, then illumination with visible light, would enable covalentcrosslinking of the thus-formed fibrin clot. This may also involvecrosslinking of the fibrin clot to other components of the extracellularmatrix (ECM) and would therefore improve the strength and stability of athrombin-induced clot for haemostasis. Fibrin is known to interact witha number of ECM proteins (Makogonenko et al (2007)

In further embodiments application of the photoactivator and electronacceptor alone, followed by irradiation, can induce crosslinking ofendogenous matrix proteins.

It is envisaged that the method of the present invention will be used toaugment or as a replacement for conventional surgical closures such assutures and staple and existing tissue adhesives generally; however, itis likely to have particular application in certain fields andapplications. In particular the method will find application in fieldswhere tissue adhesives such as fibrin glue are already used such as incardiothoracic surgery, cardiovascular surgery, thoracic surgery,hepatic and pancreatic surgery, neurosurgery, aesthetic surgery,endoscopic surgery, cranial surgery, prevention of seroma formation,bone healing, liver biopsy and dentistry.

The effectiveness of a sealant on hemostatis in cardiothoracic surgeryis important to the clinical outcome; successful local hemostatisreduces blood loss, operative time, and the need for resternotomy inthese high risk patients. Bleeding after open-heart surgery is a greatproblem in cardiac surgery. Due to hemostatic abnormalities, reoperationto control prolonged bleeding may be necessary. Therefore a sealantsuperior at producing hemostatis compared with conventional topicalagents, such as collagen-coated dressings is desirable.

The method of the present invention will also be useful for sealing airleaks from lung procedures (even as treatment for bronchopleuralfistulas). Thoracic surgery frequently involves pulmonary resection anddecortications. The consequences of such surgical intervention includehaemorrhage and air leaks. Retrospective analyses indicate thatbronchopleural fistulae occur in 2% to 3% of patients after pulmonaryresection, followed by a mortality of 15% to 20%. These complicationscan be overcome by the use of sealants of the invention.

Raw cut surfaces of soft tissues such as liver and lung cannot beisolated and secured by conventional techniques such as suturing. Themanagement of these surfaces is important for preventing intrapertonealcomplications, such as infection, abscess formation, and sepsis whichmay lead to haemorrhage, bile leakage, and fluid accumulation. Moreover,bile fluid is a severe irritant to the peritoneum and the prevention ofbile leakage using a fibrin sealant is highly desirable. Therefore thesealant of the present invention finds application as a tissue sealantin hepatobiliary surgery.

Fibrin glue is used for dural closure by neurosurgeons to preventcerebrospinal fluid leakages. The management of cerebrospinal fluid(CSF) fistulae is important. Fibrin sealant has been used inneurosurgical procedures for the prevention of CSF leakage fromfistulae, and the sealant of the present invention will find applicationin preventing CSF leakages

Aesthetic surgeons in Europe have routinely used fibrin-based glues inplace of sutures, which has enabled them to avoid the use of drains forpatients undergoing facial cosmetic surgery. There are basically twoadvantages of avoiding the use of drains and dressings: the postsurgicaltime is reduced by not putting on and removing the usual bulkydressings, and swelling, hematoma formation is reduced. Tissue adhesiveshave been reported to decrease the incidences of postoperative hematomasand edema, enable painful suture removal to be avoided, and, in somecases, facilitate early recovery and greater patient satisfaction.Plastic surgeons especially use adhesives to control burn bleeding afterdebridement and as adjuncts in surgery necessitating flaps. Skingrafting is the simplest and most effective method used to resurfacelarge burn wounds. The graft initially adheres to its new bed by a thinlayer of fibrin and nourishment of the graft occurs by plasmaticimbibition. Further ingrowth of blood vessels and fibrous tissue fromthe wound results in permanent adherence of the graft to its recipientsite known as graft “take.” This process can be hindered by collectionof blood between the graft and bed, by shearing and by infection. Theface is highly vascular and diffuse bleeding is difficult to controlfollowing burn wound excision. Traditionally, to overcome the problem ofhematoma, the grafts are meshed to enable any fluid collection to drain.Unfortunately meshing produces scarring which impairs the final cosmeticresult. Careful suturing can minimize shearing, but takes time, maypromote bleeding and also leaves scars. The sealant of the presentinvention has several advantages in the excision and skin grafting offacial burns as it provides good hemostasis and helps prevent hematomaformation, it minimizes the use of sutures, which save operating time,and it avoids further bleeding during passing of the sutures. Plasticsurgeons are also utilizing fibrin glue for the management of wrinklesof the forehead and of the aging face, and the sealant of the presentinvention will also be useful in this application. The technique avoidsthe classic coronal incision, utilized for the browlifting, thusminimizing morbidity. The adhesive not only helps to secure the foreheadand scalp flaps in place, but also works as a hemostatic agent,decreasing hematoma formation and bruising.

The collection of serous fluids after operations is a very threateningproblem and should be prevented. It can cause significant morbidity anddelayed recovery. It can appear after a mastectomy and axillarydissection, soft tissue dissection (abdominoplasty, breast reduction,facelift), and muscle harvesting. The complications include pain, woundinfection, flap necrosis, and increased costs but wound healing can beimproved with intraoperative sealant application.

Use of the tissue adhesive in bone repair should promote osteoblasticactivity rather than retarding it. In contrast, cyanoacrylates causeadverse bone reaction. Their space occupying nature prevents or retardshealing and their degradation products are harmful.

Liver biopsy is frequently necessary for candidate evaluation orhistologic follow-up of transplanted livers. Although generallyconsidered to be safe, it carries a risk of complications in up to 0.5%of cases; haemorrhage being the most important. Another option is theso-called plugged percutaneous liver biopsy (PPLB), which uses directinjection of a plugging material into the biopsy tract, and sealants ofthe present invention could be used.

In dentistry the use the use of tissue adhesives shows less propensityfor infection or delayed healing compared to the use of silk sutureswhich can result in foreign body reaction, fistula formation andsubmental abscess formation.

Tissue adhesives of the present invention may also be used as wounddressings. Absorbable adhesive bandages can be directly used in thecontrol of battlefield wounds, and immediate local control of bleedingcan be achieved. A further application may be as a hemostatic dressingin the operating room which is used instead of a sponge.

It is also envisaged that the present invention will provide a vehiclefor local administration of drugs. It has the ideal characteristics toplay such a role. In the method of the invention the thrombin is placedat the site of a tissue injury and its action there creates a matrixwhich is ultimately broken down and replaced by healing tissue as partof the body's natural healing process. Thus it initially controlsbleeding but remains firmly fixed in place until it is naturallybiodegraded. Therefore it is capable of delivery chemotactic, growthpromoting, and differentiation factors to induce both soft and hardtissue production or the innovation of undesirable proliferation. It mayalso used to deliver conventional pharmaceuticals in the form ofantibiotics and chemotherapy drugs for prolonged periods.

A wide range of drugs can be incorporated into the composition forultimate inclusion in the matrix which is formed at the site ofadministration for local action and/or systemic release. In particular,antibiotics, chemotherapeutics, peptide hormones, cytokines, antibodies,cell cycle regulators, chemokines, growth factors and secreted proteinsmay be incorporated in the matrix. The antibiotics may be from thefluoroquinolone class aminoglycocides such as hygromycin B, kanamycinand streptomycin, antifungal antibiotics such as amphotericin B,cyclohexamide, and nystatin, antineoplastic antibiotics, includingmitomycin C, puromycin, and streptozocin, antitubercular antibiotics,including rifampicin and capreomycin, lactam antibiotics such asamoxicillin and penicillin, macrolide antibiotics, including nystatinand brefelden A, peptide antibiotics, including echinomycin andgramicicdin, tetracyclines, chloramphenicol and tunicamycin. Exemplarycytokines include, but are not limited to, the interleukins,beta-interferon, alpha-interferon, gamma-interferon, angiostatin,thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, tumornecrosis factor (TNF), and bone morphogenetic proteins (BMPs).Chemokines generally act as chemoattractants to recruit effector cellsto the site of chemokine expression. Therefore the chemokines canrecruit immune system components to the site of treatment. Suitablechemokines include, but are not limited to, RANTES, MCAF, MIP1-alpha,MIP1-Beta, and IP-10. Suitable growth factors include, but are notlimited to, TGF-α, TGF-β, EGF, PDGF, FGFs, NGF, VEGF and KGF. Suitablesecreted proteins include, but are not limited to, blood factors such asFactor VIII, Factor IX, von Willebrand Factor, and the like. Anti-cancerdrugs have been demonstrated to show sustained release from a fibringlue (Yoshida et al., 2000). Fibrin glues may also provide a slowrelease formulation for antibiotics when used in ocular surgery (Maroneaet al., 1999). Furthermore fibrin glues have included antibiotics suchas amikacin to prevent local graft infection (Nishimotol et al., 2004).

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

Example 1 Photochemical Cross-Linking of Bovine Fibrinogen

A photochemical method was used to cross-link the soluble fibrinogeninto a solid biomaterial and to effect the covalent cross-linking of thefibrinogen matrix to the proteins contained in the extracellular matrixsurrounding the muscle tissue. Two small strips of bovine longissimusdorsi (LD) were dissected and the opposing surfaces coated in thesealant solution (200 mg/ml bovine fibrinogen was dissolved in PBS, with2 mM [Ru(bpy)₃]Cl₂, 10 mM ammonium persulfate). Following 10 sec ofirradiation, the two pieces of muscle were firmly attached. The lightsource chosen for the present study was a 600-W tungsten-halide source(2×300-W lamps; GE #38476). The spectral output showed a broad peak from300 nm-1200 nm. Bovine fibrinogen (Fraction I, Sigma) (200 mg/ml) wasdissolved in PBS, with 2 mM [Ru(bpy)₃]Cl₂, 10 mM ammonium persulfate)and photochemically cross-linked (600 W at 10 cm for 10 s).

Example 2 Time of Light Exposure:

Reactions contained 25 μg of bovine fibrinogen (Sigma); 2 mM[Ru(bpy)₃]Cl₂; 20 mM persulfate (Sodium salt) all in 25 μl PBS.

Reactions were exposed to 300 W incoherent light from Quartz Halogendichroic source for 1, 2, 5, 10, 30 and 60 seconds all resulted in theformation of high molecular weight, cross-linked fibrinogen polymers(FIG. 1).

Example 3

Effect of Concentration of [Ru(bpy)₃]Cl₂:

Reactions contained 25 μg of bovine fibrinogen (Sigma); 20 mM persulfate(Sodium salt) all in 25 μl PBS. Reactions were exposed to 300 Wincoherent light from Quartz Halogen dichroic source for 1 min (FIG. 2)showing that the cross-linking reaction occurs across a range of [Ru(bpy)₃]Cl₂ concentrations.

Example 4 Effect of Concentration of Persulfate:

Reactions contained 25 μg of bovine fibrinogen (Sigma); 2 mM[Ru(bpy)₃]Cl₂ (Aldrich) all in 25 μl PBS. (SPS: sodium persulfate; APS:ammonium persulfate).

Reactions were exposed to 300 W incoherent light from Quartz Halogendichroic source for 1 min (FIG. 3) demonstrating efficacy across a rangeof concentrations.

Example 5 Demonstration of Alternative Electron Acceptors (Oxidants)Cross-Linking Protein

Alternative oxidants for formation of protein hydrogels wereinvestigated in the following tests. Fibrinogen derived from bovineplasma (Sigma cat #F8630) at a concentration of 5 mg/ml was used as theprotein substrate for this investigation and was combined with thefollowing different oxidants individually at a final reactionconcentration of 10 mM.

-   -   1. Sodium Persulphate (Na₂S₂O₈),    -   2. Sodium Periodate (NaIO₄),    -   3. Vitamin B12 (C₆₂H₉₀ClCoN₁₃O₁₅P),    -   4. Ammonium cerium(IV)sulphate dehydrate (Ce(NH₄)₄(SO₄)₄. 2H₂O)    -   5. Ammonium cerium(IV) nitrate (Ce(NH₄)₂(NO3)₆    -   6. Oxalic acid (HOOCCOOH.2H₂O),    -   7. EDTA (C₁₀H₁₃N₂O₈Na₃).        Protein and oxidant were combined with 2 mM of the catalyst,        Tris(2,2″-bipyridyl)dichlororuthenium(II)hexahydrate        (C₃₀H₂₄Cl2N₆Ru.6H₂O) and immediately photoactivated for 60        seconds using a xenon 300 watt light source. Following this        reaction, 0.5 ug of reacted protein was run under denaturing        conditions on 10% BisTris SDS-PAGE. The gel was then stained        using colloidal coomassie and effects of the cross-linking        reaction determined. The results demonstrate that the oxidants;        Sodium Persulphate and Sodium Periodate work strongly in        cross-linking protein in the reaction. The other oxidants        investigated also demonstrated efficacy in protein cross-linking        but to a much lesser degree.

FIG. 4 illustrates the effects of the different oxidants oncross-linking of Fibrinogen at 5 mg/ml in PBS. In summary, the degree ofcross-linking was determined by an increase in size of the proteinrelative to the protein standards (lanes S). Lane 1 shows no proteincross-linking of the Fibrinogen only reaction. Lanes 2, 4, 7 and 9;Ruthenium only, Ruthenium plus Vitamin B12, Ruthenium plus Oxalic acidand Ruthenium plus EDTA show slight cross-linking of the Fibrinogen.Lanes 5 and 6; Ruthenium plus Cerium Sulphate and Ruthenium plus CeriumNitrate, show partial cross-linking of the Fibrinogen. Lanes 3 and 8;Sodium Persulphate and Sodium Periodate showed complete cross-linking ofthe Fibrinogen as demonstrated by the high molecular weight proteinpolymer remaining at the top of the gel.

Example 6 Demonstration of Alternative Metal Ligand Complexes(Catalysts) for Cross-Linking Protein

Two different catalysts;

-   -   1) Tris(2,2″-bipyridyl)dichlororuthenium(II)hexahydrate        (C₃₀H₂₄Cl2N₆Ru. 6H₂O) and    -   2) Hemin (C₃₄H₃₂ClFeN₄O₄)        were analysed in conjunction with the oxidants; Sodium        Persulphate (Na₂S₂O₈) and Hydrogen Peroxide (H₂O₂) for their        ability to cross-link Fibrinogen. The protein at 5 mg/ml was        combined with the catalyst at a concentration of 1 mM and        oxidant at 10 mM and immediately photoactivated using a xenon        cool light source for 60 seconds. Following this reaction, 0.5        ug of reacted protein was run under denaturing conditions on a        10% BisTris SDS-PAGE. The gel was then stained using colloidal        coomassie and effects of the cross-linking reaction determined.        These results demonstrate that either Ruthenium or Hemin can be        used as catalysts in cross-linking of proteins.

FIG. 5 illustrates the effects of the catalysts and oxidants oncross-linking of the Fibrinogen. Lane S is the protein standard. Lane 1;Fibrinogen only, shows no cross-linking. Lanes 3, 4 and 7; Fibrinogenwith Sodium Persulphate, Hydrogen Peroxide and Hemin only respectivelyshowed no cross-linking. Lanes 2 and 6; Fibrinogen plus Ruthenium andFibrinogen plus Ruthenium and Hydrogen Peroxide show slightcross-linking. Lane 5; Fibrinogen plus Ruthenium and Sodium Persulphateshows complete cross-linking of the protein as seen by the proteinremaining at the top of the gel. Lanes 8 and 9; Fibrinogen plus Heminand Sodium Persulphate and Fibrinogen plus Hemin and Hydrogen Peroxidedemonstrate partial cross-linking of the protein as demonstrated by thesmear of cross-linked protein polymer located at higher molecular weightlocations and remaining at the top of the gel.

Example 7 Photochemical Cross-Linking of Protein Solutions

A photochemical method was used to cross-link the protein solution intoa solid article and to effect the covalent cross-linking of theproteins. An appropriate concentration of protein solution (typically0.5-2% or more for collagen; typically 5% or more for other proteins,e.g. fibrinogen) in buffer solution is mixed with 2 mM Ru(Bpy)3 and 20mM persulphate salt (sodium, ammonium, potassium etc.) and irradiatedwith white light (450 nm nominal wavelength) for at least 10 secs. toform the hydrogel. The light source chosen for the present study was a600-W tungsten-halide source (2×300-W lamps; GE #38476). The spectraloutput showed a broad peak from 300 nm-1200 nm. This process is cellcompatible. To form 3-dimensional structures the article can be cast orcontained within transparent moulds.

Example 8 Casting Various Shapes Using the PICUP Cross-Linking Method

A protein solution was mixed with Ru(Bpy)₃ to 2 mM final concentrationand APS was added to 20 mM final concentration. The solution was mixedand placed into an appropriate transparent mould. The sample wasirradiated using a 600 W tungsten-halogen lamp for 10 seconds at adistance of 15 cm. The solidified protein was then removed from themould (FIGS. 9 and 10).

Example 9 Degradation of and Tissue Response to Polymerised FibrinogenBiopolymers In Vivo

The solid fibrinogen biomaterial to be evaluated in a rat subcutaneousimplant study was derived from a purified soluble fibrinogen proteinwhich was cross-linked using a photochemical method involvingTris(bipyridyl) Ruthenium (II) chloride (2 mM final concentration) andammonium persulphate (20 mM final concentration). The light sourcechosen for these studies was a 600-W tungsten-halide source (2×300-Wlamps; GE #38476). The spectral output showed a broad peak from 300nm-1200 nm.

Method Animals

40, female, 8 week old, Wistar rats were purchased from the AnimalResource Centre, Canning Vale Wash. The rats were allowed to acclimatiseto their new surroundings for 2 weeks prior to implantation offibrinogen samples.

Anaesthesia

Isoflurane, gaseous anaesthetic was used as the anaesthetic of choicebecause it has rapid induction and fast recovery. Each rat was inducedwith isoflurane (5%) in a mixture of oxygen (2 litres/minute). Inductionof anaesthesia took approximately 30-60 seconds. Once the concentrationof Isoflurane was reduced to 2% in a mixture of oxygen (2litres/minute).

Subcutaneous Implantation

The dorsum of the rat was shaved with clippers and the skin wasdisinfected with Iodine surgical scrub. A small incision was then made(approx 7 mm) through the dermis to the muscle layer. A pocket thencreated by parting the connective tissue between the dermis and musclelayer using blunt/blunt scissors. The sample plug was then gently placedin the pocket and positioned away from the initial incision point. Thewound was closed using 2 9 mm wound clips. Groups 2, 4 & 5 had 2subcutaneous biopolymers implanted per rat, each in a separate pocket.Group 3 only one biopolymer was implanted.

The samples implanted are as follows (suspended in PBS+proteaseinhibitor cocktail):

-   -   1) Fibrinogen (Sigma fraction I) (cross-linked using        photochemical method—the product is applied as a composition        comprising fibrinogen, the Ru(II) catalyst and ammonium or        sodium persulfate referred to hereinafter as “Fibrinogen-based        Tissue Sealant” or “FBTS” and then irradiated)

Each plug (100 μl of 200 mg/ml) of cross-linked fibrinogen was conicalin shape: 4 mm on base, 1 mm at top and 5-6 mm high. All plugs werebeige/brown in colour.

The height (width) and length of each polymer was measured using digitalcallipers weekly for the first 4 weeks and then every 2 weeks for theremainder of the experiment.

One week after implantation of the fibrinogen samples no swelling wasobserved (except one rat where the wound is most likely infected). Allanimals seem normal in behaviour and appearance.

Two weeks after implantation all plugs that had increased in size weresimilar in size to that implanted or had slightly reduced. Noinflammation was observed.

Three weeks after implantation 2 rats from each group were killed. Nogross pathology was noted in any organ and all organs (heart, liver,spleen, lung and kidney) were all histologically normal. Most plugsappeared to have started to degrade/be reabsorbed. Most had a thincapsule covering the plug. The fibrinogen plugs were flattened. Nomacroscopic/gross inflammation was noted at any of the implantationsites or around any of the plugs.

Eight weeks: 2 rats were killed from each group. The fibrinogen plugshad reduced in size and were spherical in shape. One animal from thefibrinogen group had hardened kidneys with an enlarged spleen—possiblecarcinoma of the kidneys, however this was unrelated to the implant.

Eighteen weeks: 2 rats from each group were killed. No plugs were seenin animals implanted Fibrinogen. The plugs had fully degraded/beenreabsorbed. There was no gross pathology seen in any of the major organsfrom all animals.

Thirty six weeks—Study Terminated. The study was terminated. Theimplanted plugs from all groups had fully degraded or been reabsorbed.No gross pathology was noted in any of the major organs from any animal.

Example 10 Photochemical Cross-Linking of Gelatin and Acid-Denatured BSA

Gelatin (Sigma) was dissolved at 65° C. in PBS at the concentrationsshown in Table 1.

BSA was dissolved in 60 mM sodium acetate pH 4.0 at room temperature ateither 20% or 10% final concentration for adhesive testing.

Ruthenium tris-bipyridyl chloride (RuBpy₃Cl) and sodium persulphate(NaS₂O₈) were prepared in sterile water at 50 mM and 500 mM,respectively. These reagents were added to final concentrations shown inTable 1.

By way of comparison, Tisseel (Duo 500 1 ml) was obtained from Baxter(NSW Australia). 100 μl of Tisseel tissue adhesive solution was added toone surface of bovine amnion and the two opposing amnion surfacesbrought together and held under light pressure for 15 min at roomtemperature prior to tensile testing.

Example 11 Tensile Testing

A perspex uniaxial tensile testing jig was constructed to measure theadhesive strength of adherent tissue surfaces. Bovine amnion wasprepared following separation of the chorion from fresh amniotic sacabattoir specimens. The pieces of amnion were cut into 5 cm×5 cm samplesand fixed over the top surface via rubber o-rings to the test jig. 100μl of tissue adhesive solution was added to one surface and the twoopposing surfaces brought together and immediately illuminated using a600 W tungsten halide light source. The light source was a 600-Wtungsten-halide lamp (2×300-W lamps; GE #38476). The spectral outputshowed a broad peak from 300 nm-1200 nm. Lead weight was addedprogressively until failure. The breaking stress was measured in kPa,and calculated using a cross-sectional area of 1.76 cm².

TABLE 1 Tissue adhesive strength Maximum Adhesive Type of denaturedprotein strength breaking (crosslinking conditions) stress (kPa)Gelatin - Bovine Type B - 15% high 92.8 bloom (Sigma G9391) 2 mMRu(Bpy)₃, 20 mM SPS Gelatin - Bovine Type B - 15% low 87.1 bloom (SigmaG6650) 2 mM Ru(Bpy)₃, 20 mM SPS Gelatin - Porcine Type A - 15% 75.2 highbloom (Sigma G2500) 2 mM Ru(Bpy)3, 20 mM SPS Gelatin - Porcine Type A -15% low 81.8 bloom (Sigma G 6144) 2 mM Ru(Bpy)₃, 20 mM SPS Gelatin -Porcine Type A - 25% low 81.5 bloom (Sigma G 6144) 2 mM Ru(Bpy)₃, 20 mMSPS Gelatin - Cold water fish skin - 81.1 40% (Sigma G7041) 2 mMRu(Bpy)₃, 40 mM SPS Gelatin - Cold water fish skin - 73.3 30% (SigmaG7041) 2 mM Ru(Bpy)₃, 40 mM SPS Gelatin - Cold water fish skin - 67.530% (Sigma G7041) 2 mM Ru(Bpy)₃, 20 mM SPS BSA 20% - acid denatured (60mM ~52 sodium acetate pH 4.0); 2 mM Ru(Bpy)₃, 20 mM SPS BSA 10% - aciddenatured (60 mM 52.7 sodium acetate pH 4.0); 2 mM Ru(Bpy)₃, 20 mM SPSTisseel ™ (Baxter) control 18.8

Table 1 shows the adhesive bond strengths of various denatured proteinsused as tissue adhesives in the current application. Data are presentedalongside adhesive strength obtained using a commercial fibrin glue(Tisseel). All of the denatured protein samples tested showed highermaximum breaking stress than Tisseel.

Example 12 Hydrolysis of Gelatin

1 mg of gelatin was dissolved in 1 ml of 6N Hydrochloric acid containing0.02% Phenol. Sample is then heated at 110° C. for 24 hours. At the endof the incubation period samples are dried and ready for thederivatisation process.

Derivatisation of Amino Acids

Reconstitute hydrolysed sample in 25 μl of Ethanol:Water:Triethylamine(2:2:1) and mix. Dry samples under vacuum at 60° C. After samples aredry add 25 μl of Ethanol: Water:Triethylamine:Phenylisocyanate (7:1:1:1)and mix. Incubate the samples at room temperature for 30 minutes andthen dry under vacuum at 60° C. The samples are then reconstituted in500 μl Mobile phase A ready for HPLC analysis.

High Performance Liquid Chromatography of Amino Acids Conditions Column:Phenomenex BF 4252-EO, Luna 5 μm C18 (2), 150×6.0 mm. Wavelength: 254 nmOven Temperature: 40° C. Mobile Phase A: 0.14M Sodium Acetate, 0.05%Triethylamine, pH 6.5 Mobile Phase B: 60% Acetonitrile Injection Volume:25 μl

TABLE 2 Amino acid composition (in mol % or Area %) of gelatin (bovineskin, type B) not crosslinked or crosslinked using photochemicalcrosslinking described above. Area % Gelatin, Area % Ru, Ru Gelatin,Amino Area % not Ru, Ru Acid Mol %* Gelatin leached leached† Asp 2.8 3.32.3 2.8 Thr 1.9 1.4 1.4 1.4 Ser 2.8 3 3.1 2.9 Glu 7.6 6.1 5.7 5.4 Pro12.3 15.2 15.1 14.9 Gly 32.3 31.1 34.2 32.7 Ala 14.2 11.1 11.3 10.9 Val2.2 2 2.1 2.0 Cys — — — — Met 0.9 0 0.2 0 Ile 1.9 1.2 1.1 2.2 Leu 2.82.3 2.1 2.1 Tyr 0.9 0.3 0 0 Phe 1.9 1.4 1.4 1.4 Lys 2.8 5.2 5.7 8.7 His0.9 0.4 0.4 0.4 Arg 3.8 4.7 4.2 2.4 OH-Pro 6.6 11 9.9 10.8 OH-Lys 0.9 —— —

Table 2 shows the amino acid composition of bovine gelatin measuredbefore and after photochemical crosslinking. The loss of a measurabletyrosine peak in the crosslinked sample supports the role of tyrosine inthe crosslinking mechanism.

Example 13 Stabilisation of Gelatin Beads

Beads were made from 25% w/v A-type gelatin (175 g Bloom) heated to 50 Cto dissolve. After cooling to 37° C., sodium persulfate (10 mM finalconcentration) and tris bipyridyl ruthenium (2 mM final concentration)were added in the dark and the mixture dispersed by addition with an 18Gneedle at 10% v/v in olive oil at 50° C. by rapid stirring, whilemaintained in the dark. After 30 min, the emulsion was transferred to20° C. and illuminated while stirred twice for 2 min. at 15 minintervals and then every 30 min for a further 5 hours using a 500 Wquartz-halogen lamp. Beads were separated by sedimentation followed byextraction with ethanol and/or acetone. The effective stabilisation ofthe beads was shown after rehydration and addition to water at 56° C.After 16 hr, no dissolution nor shape changes of the beads was observed.

Example 14

Delivery of cross linked protein using a sponge support. Fibracolcollagen sponge impregnated with 15% bovine gelatin (with Ru(Bpy)3 andSPS)

Method

The adhesive strength of photochemically cured gelatin, delivered in asponge was assessed by impregnating a 176 mm² disc of Fibracol Pluscollagen sponge (Ethicon) with 500 μl of 15% Bovine gelatin (SigmaG9391) dissolved in PBS. The adhesive bond strength of this formulationwas measured using a tensile testing jig to assess the adhesive strengthto bovine amnion membrane.

The gelatin solution was maintained at 45° C. in a water bath, the[Ru(Bpy)₃]²⁺ was added to 2 mM and sodium persulphate added to 20 mMfinal concentration. The solution was mixed thoroughly and the FibracolPlus sponge membrane then thoroughly impregnated with the proteinmixture. The soaked membrane was then placed between the amnionmembranes and the upper half of the jig was lowered to meet the lowerhalf with a small force (˜250 gf) applied. The assembled test jig wasilluminated for 60 seconds using a 300 W xenon lamp. Samples were testedfor tensile stress at break in triplicate.

Results 1. 15.4N 2. 12.95N 3. 10.72N Mean=13.02N/176 mm²

Maximum tensile stress at break=73.7 kPa

These data demonstrate that photochemically cross-linked gelatin can bedelivered using an inert carrier such as a collagen sponge.

Example 15 Tensile Testing of Photochemically-Cross-Linked Fibrinogen

Tensile tests were carried out on cross-linked fibrinogen inphosphate-buffered saline (PBS) buffer on an Instron Tensile Tester(model 4500) at a rate of 5 mm/min and a temperature of 21° C. Theswollen dumbbell-shaped strip samples (30 mm×4 mm×1 mm) had a gaugelength of 8 mm and strain was increased until failure occurred (FIG.19). The elastic modulus (E) was measured at 20%, 40% and 50% strain,yielding figures of 77 kPa, 85 kPa and 87 kPa respectively (FIG. 20).These measurements of Young's Modulus are similar to data obtained fromstudies using two commercial fibrin-based tissue sealants. The extensionto break (Eb) was 135% and the ultimate tensile strength UTS was 141kPa. Velada et al (2002) compared the mean tensile strength of severalcommercial fibrin sealants (Vivostat, Tussucol and Beriplast) and thesewere found to be in the range 38 kPa to 55 kPa.

The resilience of the cross-linked fibrinogen hydrogel was determined at10% and 20% strain and yielded a figure of 70.7%, considerably less thanresilin (97%), but illustrating that cross-linked fibrinogen hydrogelsconsist of elastic domains. Importantly, the extension to break was135%, illustrating the extensibility of the photochemically cross-linkedfibrinogen biomaterial. Velada et al reported the mean extension tobreak of commercial fibrin sealants to be 103%±13% but tensile strengthvaried by 2-5-fold with fibrinogen concentrations in the range 25-100mg/ml.

Example 16 In Vivo Study of Fibrinogen Scaffold

Porous hydrogel scaffolds of photo-crosslinked fibrinogen, seeded withcells, were implanted subcutaneously into nude mice. The viability ofthe implanted cells and integration of the scaffolds with surroundingtissue were assessed at 2 and 4 weeks after implantation of thescaffolds.

Materials and Methods

The scaffolds contained 60 mg/ml bovine fibrinogen in Dulbecco'sModified Eagle's Medium. The scaffolds also contained 50 μg/ml bovinecatalase and 1% hydrogen peroxide to induce foaming and hence produce aporous matrix, and 2 mM ruthenium and 20 mM sodium persulfate to achievephoto-crosslinking during exposure to blue light for 30 s. The scaffoldswere seeded with 2×10⁶ cells/ml of C2C12 mouse myoblasts. Thecell-containing scaffolds were cultivated for three days in vitro, thensurgically implanted into 8 week old nude mice. Two implants were placedsubcutaneously in each animal, one on either side of the mid-dorsalline. Animals were sacrificed at 2 weeks (three animals) and 4 weeks(two animals) post-surgery and the scaffolds and surrounding tissue wereremoved. Samples were examined macroscopically and histologically.

Results

At 2 and 4 weeks post-surgery, the implanted scaffolds were wellintegrated into surrounding tissue and all organs were normal.Histological examination showed that the implanted C2C12 myoblasts hadsurvived and proliferated, as clearly evidenced by the differentiationof several myoblasts into multinucleated, thickened and elongatedmyotubes. There was also microscopic evidence of the integration ofmultiple new blood vessels into the scaffold (FIG. 22).

This example demonstrates that scaffolds containing cells have beensuccessfully implanted into nude mice, with evidence of survival,proliferation and differentiation of the originally implanted cells.There is also clear evidence of integration of the scaffolds withsurrounding tissue as well as vascularisation of the implantedscaffolds.

Example 17 Stabilization of a Thrombin Induced Clot by CrosslinkingUsing a Photochemical Method

Fibrinogen (Sigma Fraction1) was dissolved at either 5 mg/ml or 50 mg/mlin phosphate-buffered saline (Dulbecco's PBS without Ca & Mg). Thrombin(Sigma—from bovine plasma, 34.8 U/mg solid) was prepared as a 20 mg/mlsolution in PBS. Ruthenium tris-bipyridyl chloride (RuBpy₃Cl) and sodiumpersulphate (NaS₂O₈) were prepared in sterile water at 50 mM and 500 mM,respectively.

FIG. 21 shows the result of treating two concentrations of fibrinogenfor 2 minutes at room temperature with thrombin. Panel A shows a clotformed from a 5 mg/ml solution of fibrinogen (similar to theconcentration of fibrinogen in blood—ref: Weisel J W. Fibrinogen andfibrin. Adv Protein Chem. 2005; 70:247-99). Panel B shows a stiffer clotformed from a 50 mg/ml solution of fibrinogen. Both fibrinogen solutionswere treated with 10.5 U of thrombin at room temperature. Both clotswere completely soluble in 2.5% acetic acid within 2 minutes at roomtemperature. Panel C shows photochemically crosslinked fibrin (samplestreated as in A, but 2 mM ruthenium tris-bipyridyl and 20 mM sodiumpersulphate added simultaneously with thrombin in the dark). The sampleswere then illuminated with white light (600 W tungsten halide lamp) for10 seconds. Samples were subsequently soaked in 2.5% acetic acid (“5” isfibrinogen at 5 mg/ml; “50” is fibrinogen at 50 mg/ml) and wereinsoluble as shown. Panel D shows a fibrinogen sample (5 mg/ml) treatedwith 2 mM ruthenium tris-bipyridyl and 20 mM sodium persulphate, addedsimultaneously with thrombin in the dark. The fibrin clot wassubsequently transferred in the dark to a solution of 2.5% acetic acid.After 2 minutes at room temperature, the clot dissolved completely,demonstrating that, without illumination, no covalent crosslinkingoccurred in the fibrin clot.

The data (FIG. 21) demonstrates that following visible lightillumination via a photochemical reaction, addition of rutheniumtris-bipyridyl and sodium persulphate to thrombin stabilizes the clotformed from fibrinogen. This reaction is independent of any action ofFactor XIII. A clot formed via the action of thrombin, in vivo, wouldsimilarly be covalently crosslinked via this photochemical process andthat this clot will be covalently bonded to the protein components inthe ECM, thus forming a more robust clot at the wound site.

REFERENCES

The disclosure of the following documents is incorporated herein byreference:

-   Barnes C P, Smith M J, Bowlin G L, Sell S A, Tang T, Matthews J A,    Simpson D G, Nimtz J C “Feasibility of Electrospinning the Globular    Proteins Hemoglobin and Myoglobin” Journal of Engineered Fibers and    Fabrics Vol 1 No. 2, 16-29(2006)-   Brown, K C and Kodadek, T Met Ions Biol Syst. 2001; 38:351-84.    “Protein cross-linking mediated by metal ion complexes”-   Dickneite, G., H. J. Metzner, M. Kroez, et al. “The Importance of    Factor XIII as a Component of Fibrin Sealants.” Journal of Surgical    Research 107 (October 2002): 186-195.-   Dodd, R. A., R. Cornwell, N. E. Holm, et al. “The Vivostat    Application System: A Comparison with Conventional Fibrin Sealant    Application Systems.” Technology and Health Care 10 (2002): 401-411.-   D A. Fancy and T. Kodadek “Chemistry for the analysis of    protein-protein interactions: Rapid and efficient cross-linking    triggered by long wavelength light.” Proc. Natl. Acad. Sci. Vol. 96,    pp. 6020-6024, May 1999-   David A Fancy, Carilee Denison, Kyonghee Kim, Yueqing Xie, Terra    Holdeman, Frank Amini and Thomas Kodadek “Scope, limitations and    mechanistic aspects of the photo-induced cross-linking of proteins    by water-soluble metal complexes” Chemistry & Biology (2000)    7:697-708-   Furst W, Banerjee A, Redl H. Comparison of structure, strength and    cytocompatibility of a fibrin matrix supplemented either with    tranexamic acid or aprotinin. J Biomed Mater Res B Appl    Biomater. (2007) 82:109-14-   Jackson, M. R. “Fibrin Sealants in Surgical Practice: An Overview.”    American Journal of Surgery 182 (August 2001) (2 Suppl): 1S-7S.-   Kodadek T, Isabelle Duroux-Richard and Jean-Claude Bonnafous,    “Techniques: Oxidative cross-linking as an emergent tool for the    analysis of receptor-mediated signalling events” TRENDS in    Pharmacological Sciences Vol. 26 No. 4 Apr. 2005-   Khadem, J., Veloso, A. A., Tolentino, F. T., Hasan, T. and    Hamblin, M. R., “Photodynamic Tissue Adhesion with Chlorin_(e6),    Protein Conjugates”. IOVS, December 1999, Vol. 40, No. 13.-   Lee, K-C, Park, S-K and Lee, K-S (1991) neurosurgical applications    of fibrin sealants. 9^(th) Annual congress of the world society of    cardio-thoracic surgeons; November 1999, Lisbon, Spain.-   Makogonenko E, Ingham K C, Medved L. Interaction of the fibronectin    COOH-terminal Fib-2 regions with fibrin: further characterization    and localization of the Fib-2-binding sites. Biochemistry. (2007)    May 8; 46(18):5418-26. Epub 2007 Apr. 11-   Mankad, P. S., and M. Codispoti. “The Role of Fibrin Sealants in    Hemostasis.” American Journal of Surgery 182 (August 2001) (2    Suppl): 21S-28S.-   Maronea Piero, Monzillob Vincenza, Segua Catia, Antoniazzic Elena,    “Antibiotic-Impregnated Fibrin Glue in Ocular Surgery: In vitro    Antibacterial Activity”, Ophthalmologica 1999; 213:12-15.-   Matras, H (1985) Fibrin seal: the state of the art. J Oral    Maxillofac Surg 43: 605-611.-   McManus, M, Sell S A, Espy P G, Koo, H P and Bowlin G L (2006) “On    the Road to in situ Tissue Regeneration: A Tissue Engineered    Nanofiber Fibrinogen-Polydioxanone Composite Matrix” Proceedings of    Mid-Atlantic section of the American Urological Association Annual    Meeting, 2006 www.maaua.org/abstracts/2006/07.cgi-   Milne, A A, Murphy, W G, Reading, S J and Ruckley, C V (1995) Fibrin    sealant reduces suture line bleeding during carotid endarterectomy:    a randomised trial. Eur J Endovasc Surg 10: 91-94-   Morikawa, T. “Tissue Sealing.” American Journal of Surgery 182    (August 2001) (2 Supply: 29S-35S.-   Mosesson M W. Fibrinogen and fibrin structure and functions. J    Thromb Haemost. (2005) 3:1894-904;-   Mosesson M W, Siebenlist K R, Meh D A. The structure and biological    features of fibrinogen and fibrin. Ann N Y Acad Sci. 2001;    936:11-30).-   Nishimotol Kazuo, Yamamura Keiko, Fukase Fumiaki, Kobayashi)    Masayoshi, Nishikimil Naomichi and Komoril Kimihiro, “Subcutaneous    tissue release of amikacin from a fibrin glue/polyurethane graft”,    Journal of Infection and Chemotherapy; Vol. 10, No. 2 (2004) pages    101-104.-   Velada J L, Hollingsbee D A, Menzies A R, Cornwell R, Dodd R A.    Reproducibility of the mechanical properties of Vivostat system    patient-derived fibrin sealant. Biomaterials. 2002 May;    23(10):2249-54.-   Yoshida H, Yamaoka, Y., Shinoyama M., Biol Pharm Bull. 2000; pages    371-374 “Novel drug delivery system using autologous fibrin    glue-release properties of anti cancer drugs”, Department of    Pharmacy, Yamaguchi University Hospital, Ube, Japan.

1-29. (canceled)
 30. A biomaterial comprising a 3-dimensional matrix ofa protein or peptide crosslinked through irradiation a photoactivatablemetal-ligand complex and an electron acceptor in the presence of theprotein or peptide, thereby initiating a cross-linking reaction to forma 3-dimensional matrix of the biomaterial.
 31. A biomaterial as claimedin claim 30 wherein the biomaterial is shaped to form a prosthesis. 32.A biomaterial as claimed in claim 30 wherein the biomaterial is in theform of a sheet or mat.
 33. A biomaterial as claimed in claim 30 whereinthe biomaterial is a membrane, sponge or foam.
 34. A biomaterial asclaimed in claim 30 wherein the biomaterial is produced in the form offibers or beads.
 35. A biomaterial as claimed in claim 30 wherein thebiomaterial is a scaffold.
 36. A biomaterial as claimed in claim 30wherein the biomaterial further comprises one or more agents selectedfrom the group consisting of: cells, growth factors, bioactive agents,drugs, therapeutic agents, and nutrients. 37-52. (canceled)
 53. Abiomaterial as claimed in claim 30, wherein the protein or peptidecomprises an at least partially denatured protein.
 54. A biomaterial asclaimed in claim 53, wherein denaturation of the protein or peptideoccurs as the result of application of heat, physical or mechanicalagitation, acid/alkali treatment, or as a result of chemicalmodification.
 55. A biomaterial as claimed in claim 53, wherein the atleast partially denatured protein comprises fibrinogen, fibrin,collagen, fibronectin, keratin, laminin, serum albumin, or admixturesthereof.
 56. A biomaterial as claimed in claim 53, wherein the at leastpartially denatured protein comprises gelatin.
 57. A medical implantstructure comprising: a biomaterial comprising a crosslinked3-dimensional matrix of a protein or peptide; and a substrate, whereinsaid biomaterial is coated on a surface of said substrate.
 58. A medicalimplant structure as claimed in claim 57, wherein said substrate is atissue portion.
 59. A medical implant structure as claimed in claim 57,wherein the medical implant structure is in the form of a sheet.
 60. Amedical implant structure as claimed in claim 57, wherein thebiomaterial further comprises one or more agents selected from the groupconsisting of: cells, growth factors, bioactive agents, drugs,therapeutic agents, and nutrients.
 61. A medical implant structure asclaimed in claim 57, wherein the protein or peptide comprises an atleast partially denatured protein.
 62. A medical implant structure asclaimed in claim 61, wherein denaturation of the protein or peptideoccurs as the result of application of heat, physical or mechanicalagitation, acid/alkali treatment, or as a result of chemicalmodification.
 63. A medical implant structure as claimed in claim 61,wherein the at least partially denatured protein comprises fibrinogen,fibrin, collagen, fibronectin, keratin, laminin, serum albumin, oradmixtures thereof.
 64. A medical implant structure as claimed in claim61, wherein the at least partially denatured protein comprises gelatin.65. A medical implant structure as claimed in claim 57, wherein saidcrosslinked 3-dimensional matrix is formed by irradiation of aphotoactivatable metal-ligand complex and an electron acceptor in thepresence of the protein or peptide, thereby initiating a cross-linkingreaction.